The objective of this application is to develop the career of Dr. Alissa Weaver as an independent investigator in the field of cancer invasion and metastasis. Cortactin is a prominent src kinase substrate that is overexpressed in 13% of breast cancers via 11q13 amplification, a chromosomal change associated with increased tumor aggressiveness and poor patient prognosis. The nucleation of new actin filaments by the Arp2/3 complex is essential for protrusion of the leading edge of migrating cells and also contributes to the creation of additional subcellular structures, such as cancer cell invadopodia. Previous studies by this investigator have demonstrated that cortactin promotes actin assembly by the Arp2/3 complex and works in concert with N-WASp, another Arp2/3 activator and src substrate. Both cortactin and N-WASp have been implicated in cancer cell invadopodia formation, but the mechanism of recruitment and action is unclear. The goal of this proposal is to test the hypothesis that cortactin functions as an integrator of signals to the cytoskeleton and controls critical steps in breast cancer invasion and metastasis. Three specific aims are proposed: In Specific Aim 1, we will test the hypothesis that a key function of cortactin is to assemble signaling and cytoskeletal proteins, including src kinase and N-WASp, for actin assembly in breast cancer cell invadopodia and lamellipodia. In Specific Aim 2, we will determine the role of cortactin and N-WASp in the morphologic and phenotypic changes that characterize the epithelial-mesenchymal transition. In Specific Aim 3, we will develop a tetracycline-inducible transgenic mouse model to test the hypothesis that cortactin promotes branching morphogenesis of the mammary gland and breast cancer metastasis. We anticipate that these studies will yield important insight into the mechanisms by which metastasis occurs in vivo and potentially allow the identification of novel cytoskeleton-based targets for rational drug design.